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EP-4735605-A2 - MRP VARIANTS IN BRASSICA

EP4735605A2EP 4735605 A2EP4735605 A2EP 4735605A2EP-4735605-A2

Abstract

Provided are plants, cells, tissues, and germplasm thereof comprising one or more targeted alterations of the genomic sequences of the MRP1 or MRP2 genes that encode for multidrug resistance-associated protein ( MRP ), as well as meal made from such plant materials. Also provided are methods and compositions to make altered MRP1 and MRP2 gene variants; breeding methods and methods of identifying and selecting plant materials having the disclosed MRP1 and MRP2 variants.

Inventors

  • BETTIS, Janel M
  • KREJSA, Beverly
  • PATTERSON, THOMAS G
  • RIPP, KEVIN G
  • SHI, JINRUI

Assignees

  • Pioneer Hi-Bred International, Inc.

Dates

Publication Date
20260506
Application Date
20240626

Claims (20)

  1. 1. A method of increasing inorganic phosphate in Brassica napus plant, seed, tissue or germplasm thereof, the method comprising introducing a targeted alteration to the sequence of one or more multidrug resistance-associated protein (MRP) genes MRP1, MRP2, or both MRP1 and MRP2 and thereby generating a Brassica napus plant, seed, tissue or germplasm thereof comprising one or more MRP variants and an increased level of inorganic phosphate relative to the plant, seed, tissue or germplasm thereof prior to introducing the one or more MRP variants.
  2. 2. The method of claim 1, comprising introducing a targeted alteration of one or more alleles of MRP2 on chromosome A09 or C09 or introducing a targeted alteration of one or more alleles oiMRPl on chromosome A10 or C05.
  3. 3. The method of claim 1 or 2, wherein the targeted alteration is an insertion or deletion that reduces or eliminates expression of the full-length protein encoded by the targeted MRP gene alleles.
  4. 4. The method of claim 3, wherein the targeted alteration introduces a premature termination codon in the one or more MRP variants.
  5. 5. The method of claim 1, wherein the targeted alteration introduces a premature termination codon in the position shown in Table 2 for MRP2 on chromosome A09 or C09, Table 3 for MRP1 on chromosome A10 or C05, or Table 4 for MRP2 on chromosome A09 or C09.
  6. 6. The method of claim 1, wherein each of the one or more MRP variants comprises a premature termination codon or splice donor shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO 25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO 32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO 79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO 82, or SEQ ID NO:83.
  7. 7. The method of any one of claims 1 -6, wherein the method comprises introducing double homozygous variants of MRP 1 or MRP 2 and thereby generating Brassica napus plant, seed, tissue or germplasm thereof comprising double homozygous variants of MRP 1 oxMRP2.
  8. 8. A method of producing high inorganic phosphate Brassica napus meal, the method comprising a. selecting Brassica napus seed or grain comprising one or more MRP variants, wherein the variants comprise a targeted alteration of MRP 1, MRP2, or both MRP1 and MRP2 and the selected seed or grain comprise increased inorganic phosphate relative to control seed or grain lacking the one or more MRP variants; b. milling the selected seed or grain to produce high inorganic phosphate Brassica napus meal.
  9. 9. The method of claim 8, wherein the selected seed or grain comprises a targeted alteration to one or more alleles of MRP2 on chromosome A09 or C09 or a targeted alteration to one or more alleles of MRP 1 on chromosome A10 or C05.
  10. 10. The method of claim 9, wherein the selected seed or grain comprises an insertion or deletion that reduces or eliminates expression of the full-length protein encoded by the targeted MRP genes.
  11. 11. The method of claim 10, wherein the insertion or deletion introduces a premature termination codon in the one or more MRP variants.
  12. 12. The method of claim 11, wherein the targeted alteration introduces a premature termination codon in the position shown in Table 2 forMRP2 on chromosome A09 or C09, Table 3 for MRP1 on chromosome A10 or C05, or Table 4 for MRP2 on chromosome A09 or C09.
  13. 13. The method of claim 8, wherein the one or more MRP variants comprise one or more of the premature stop codons or splice variant shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO 32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO 62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO 65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID N0:71 , SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID N0:81, SEQ ID NO:82, or SEQ ID NO:83, including combinations thereof.
  14. 14. The method of any one of claims 8-13, wherein the selected seed or grain comprises double homozygous variants A MRP1 oxMRP2.
  15. 15. The method of any one of claims 8-14, wherein the selected seed or grain further comprises a targeted alteration of one or more of the following genes: LPA1, TT2, TT8, DFR, F3H, ANR, LDOX, MYB28 or MAM 1.
  16. 16. Brassica napus plant, seed, tissue or germplasm thereof comprising one or more multidrug resistance-associated protein (MRP) gene variants which are single homozygous MRP2 variants on chromosome A09 or C09; double homozygous MRP2 variants on chromosomes A09 and C09, single homozygous MRP1 variants on chromosome A10 or C05; or double homozygous MRP1 variants on chromosomes A10 and C05, wherein the Brassica napus plant, seed, tissue or germplasm thereof comprising the one or more MRP variants have an increased level of inorganic phosphate relative to a control lacking the one or more MRP variants.
  17. 17. The Brassica napus plant, seed, tissue or germplasm thereof of claim 16, which comprises a combination of (i) single homozygous MRP2 variants on chromosome A09 or C09 and single homozygous MRP1 variants on chromosome A10 or C05, (ii) double homozygous MRP2 variants on chromosome A09 and C09 and single homozygous MRP1 variants on chromosome A10 or C05; or (iii) single homozygous MRP2 variants on chromosome A09 or C09 and double homozygous MRP1 variants on chromosome A10 and C05.
  18. 18. The Brassica napus plant, seed, tissue or germplasm thereof of claim 16 or 17, wherein each of the MRP variants comprises a premature termination codon in the position shown in Table 2 foxMRP2 on chromosome A09 or C09, Table 3 MRP 1 on chromosome A10 or C05, or Table 4 fox MRP 2 on chromosome A09 or C09.
  19. 19. The Brassica napus plant, seed, tissue or germplasm thereof of any one of claims 16 or 17, wherein each of the one or more MRP variants comprises a premature stop codon or splice variant shown in SEQ ID NO :22, SEQ IDNO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID N0:41, SEQ ID NO:42, or SEQ ID NO:43, SEQ ID NO 53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID N0:81, SEQ ID NO:82, or SEQ ID NO: 83.
  20. 20. The Brassica napus plant, seed, tissue or germplasm thereof of any one of claims 16-19, further comprising a targeted alteration of one or more of the following genes: LPA1, TT2, TT8, DFR, F3H, ANR, LDOX, MYB28 or MAM 1.

Description

MRP VARIANTS IN BRASSICA REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY [0001] The official copy of the sequence listing is submitted electronically as an xml- formatted sequence listing file named 96770-US-PSP ST26 created on June 29, 2023, and having a size of 267,241 bytes which is filed concurrently with the specification. The sequence listing comprised in this xml-formatted document is part of the specification and is herein incorporated by reference in its entirety. FIELD OF THE DISCLOSURE [0002] The disclosures relates to phosphorous metabolism in Brassica, variants of MRP genes that reduce phytate and increase inorganic phosphate in Brassica grain and meal made thereof. BACKGROUND [0003] Brassica napus (also referred to herein as canola or oilseed rape) is an allotetraploid (2n= 4x = 38, AACC) comprising two full genome sets, four component genomes, and total of 38 chromosomes. The A genome includes 10 chromosomes and is derived from B. rapa (2n = 2x = 20, AA). The C genome includes 9 chromosomes and is derived from B. oleracea (2n = 2x = 18, CC). B. napus is one of the most important vegetable oilseed crops in the world, especially in China, Canada, the European Union and Australia. Canola meal, the fraction of the seed remaining after crushing and oil extraction, is approximately 55% of the volume of canola seed. [0004] While canola meal is rich in protein and capable of providing a substantial amount of energy when used in animal feed; it also includes levels of anti -nutritional factors such as glucosinolates, tannins, phytate (or phytic acid), and sinapine. Since meal comprises about half of the seed volume of canola, and the demand for canola/oilseed rape has risen and is expected to continue rising to meet demands for healthy cooking oils, biodiesel, and personal care products, there is a long-felt need to modify the compositional properties of canola meal and thereby increasing its nutritional value. [0005] Animal growth and development require phosphorus. While plant-based animal feed such as canola meal contains a large amount of phosphorus, most of the phosphorus is in the form of phytate. Phytate sequesters phosphate in a form that prevents its absorption by monogastric animals such as pigs and poultry. To supply monogastric animal need for free, inorganic phosphorus, plant-based meals are often supplemented with dicalcium/monocalcium phosphate or treatment with a phytase enzyme that converts phytate to usable inorganic phosphate (Pi). Such supplementation adds to the cost and steps of preparing a diet for the animals. Accordingly there is a desire for new varieties of canola that contain higher levels of inorganic phosphate (Pi) in the seeds/grain used for feed formulation. This would reduce or eliminate the need for dicalcium/monocalcium phosphate or phytase supplements, and thereby reduce the cost of feed for animal production. [0006] Biosynthetic pathway genes involved in phytate production and transport have been identified in the model plant Arabidopsis and multiple crop plants including AtITPKI, AIITPK2, OsMIPS, OsIPK, OsMIK, 0sMRP5, OsPLDl, OsLPAl, ZmMRP4, BnPGK2, BnITPKI and BnITPK . For example, maize I pal gene (ZmMRP4) seed-specific silencing was found to reduce phytate and increase Pi in maize and a similar phenotype also was observed in transgenic soybean with the expression of homologous MRP genes silenced. See Shi et al., 2007 and US7511198B2. However, in Brassica napus, BnA9.MRP5 was associated with reduced phytate inasmuch as BnA9.MRP5 transcription levels were found to be “significantly elevated” relative to a high phytate variety. Liu et al. 2021 Authorea, DOI: 10.22541/au. 161165160.08519096/vl, see Abstract. In another study, double mutants of BnMRP5 on chromosomes A10 and C09 showed “no significant differences in Pi” or inorganic phosphate. Sashidhar et al. 2020 Brontiers in Plant Science, 11 (Article 603): 1-10, at 1 and 5, 2nd col. [0007] There is a desire to modify genes and develop new varieties of Brassica napus that not only provide reduced phytate content but also higher levels of inorganic phosphate (Pi). SUMMARY OF THE DISCLOSURE [0008] The disclosed compositions and methods are based, at least in part, on the surprising discovery that levels of inorganic phosphate in Brassica napus can be increased by a targeted alteration of the genomic sequences of MRP1 and MRP2 genes that encode for multidrug resistance-associated protein (MRP'). The evidence herein demonstrates that targeted alterations of MRP1, MRP2, or both MRP1 and MRP2 lead to significantly increased levels of free or inorganic phosphate in Brassica napus plants. The term Brassica napus as used herein includes crop varieties of the species such as spring oilseed rape, winter oilseed rape, and low erucic cultivars of the foregoing which are called canola. [0009] Provided herein is a method of increasing inorganic phosphate in Brassica napus plant, cell, seed, tissue or germplasm thereof,